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Researchers using ever more sophisticated methods to look into the brain are getting a glimpse of the elusive process of memory formation. Two studies out in the current issue of the Journal of Neuroscience tackle different aspects of this same enduring problem and come up with some illuminating results. Together, the studies give a new look at the processes underlying memory formation, from the first activation of neurons by new information, to the hardwiring of that information in the form of new synapses. Understanding how neurons make memories is important to understand the undoing of that process, the reversal that caused Alois Alzheimer’s patient Auguste D. famously to proclaim, “I’ve lost myself.”

The first report, from Gary Lynch’s lab at the University of California at Irvine, uses a biochemical marker of long-term potentiation to identify individual dendritic spines that are rapidly altered in vivo as rats acquire new information. The researchers’ ability to pick out the few spines that change among the many that do not could be the first step in mapping a complete memory circuit, or engram, the long-time goal of memory researchers.

The second paper combines time-lapse microscopy in living cells with ultrastructural electron microscopy to get a new look at how the early growth of spines, like that seen in the Lynch study, leads to the formation of new synapses. Tobias Bonhoeffer and Valentin Nagerl at the Max Planck Institute of Neurobiology in Muchen-Martinsried, along with colleagues at the University of Zurich in Switzerland, show that activity-dependent synaptogenesis is an extended process where rapid spine extension leads to new contacts between cells within tens of minutes. Then, they show, that contact matures into a new synapse more slowly, over hours.

Stimulation, Spines, Synapses
Long-term potentiation is at the root of synaptic changes that lead to memory formation (see ARF related news story). Previous work from the Lynch lab showed that inducing LTP by electrical stimulation in hippocampal slices was accompanied by a dramatic and transient increase in the phosphorylation of the actin binding protein cofilin in the potentiated synapses (Chen et al., 2007). In the new paper, first author Vadim Fedulov used cofilin phosphorylation as a biochemical marker of LTP induction in vivo. They introduced rats to a new environment, where the animals engaged in exploration of new objects in an unsupervised learning paradigm. After that, the animals were sacrificed and the investigators looked for cofilin phosphorylation in the hippocampus.

Animals that had completed the learning drill showed a 30 percent increase in the number of spines that contained detectable phospho-cofilin (pCofilin), localized in the spine heads. Though the increase was robust, the labeled spines were rare—just one out of roughly 300 spines in a given brain volume were affected. Consistent with their previous work in hippocampal slices, the pCofilin-positive synapses were associated with larger post-synaptic densities, indicating morphological change occurred in a select population of synapses. The link to memory was strengthened by the demonstration that giving rats an NMDA receptor antagonist before the learning phase both prevented memory formation and the appearance of pCofilin-positive synapses.

“The links between pCofilin and LTP on the one hand, and LTP with memory on the other, suggest that the effects observed in the present study are directly related to the encoding of information,” the authors conclude. The goal of mapping a memory, making a synapse by synapse trace, will require even more powerful techniques, they concede. The ability to look at larger fields in more animals, and techniques to look at later events will need to be developed.

Notably, Lynch and coworkers did not see any increase in the total number of synapses within 30 minutes after learning. This is consistent with the work of Nagerl and Bonhoeffer, published in the same July 25 issue of the Journal of Neuroscience, showing that synaptic formation after LTP induction in hippocampal slices takes nearly a day.

In that study, the investigators filled single CA1 neurons with a double label of calcein dye and the electron-dense marker biocytin, and then subjected the cells to electrical theta-burst stimulation. After imaging the living preparations to track spine growth, they fixed the slices, and re-identified the same cells in sections for analysis of synaptic structure by EM.

As expected, they saw that electrical activity induced new spines. Interestingly, the induced spines appeared very stable, and most persisted in the daylong experiment. The new spines were clearly in contact with presynaptic boutons within tens of minutes of their budding, but there was no evidence that synapses had formed until the spines were 15-19 hours old. “The timeline of contact formation between spines and synaptic boutons in our experiments suggests a prolonged, multistep process for synaptogenesis,” the authors write. The process resembles models for developmental synapse formation. In agreement with this, the young spines mostly formed contacts with boutons that already had synapses with other targets, while later on, most spines contacted boutons with only single synapses. The authors speculate that removal of the competing synapses would complete the rewiring process.—Pat McCaffrey